Ovarian endocrine cells form functional syncytia, due to extensive intercellular gap junctional (GJ) communication. Identification of mechanisms underlying intragonadal GJ communication may be a key to the understanding of ovarian physiology. The relative paucity of information concerning the protein makeup of GJs in the primate ovary prompted us to identify the Cx type(s) expressed in the rhesus monkey and human ovary and to define the cellular sites of their synthesis. In immunohistochemical studies we used specific antisera against Cx43, Cx32, and Cx26. There was strong staining for Cx43 in thecal and interstitial cells, GCs and luteal cells of the monkey and human ovary. No Cx26 staining was detected. While Cx32 immunoreactivity was present in thecal cells, it was noted only in a subpopulation of GCs in both human and monkey ovaries. Western blots revealed that while Cx43 protein was abundant in luteinized human GCs obtained from IVF patients, Cx26 was absent, and Cx32 was present in small amounts. Immunocytochemical staining of human cultured GCs showed strong Cx43 immunoreactivity at the contact zones of virtually every cell, but Cx32 immunoreactivity was seen in only some of the cells. RT-PCR of monkey ovarian mRNA and human luteinized GC mRNA demonstrated that the Cx32 gene is indeed expressed in the monkey ovary and human GCs. Using the monkey Cx32 PCR fragment as a probe for in situ hybridization experiments, Cx32 mRNA was detected in thecal cells and in a subpopulation of GCs. The presence of two Cx subtypes, Cx43 and Cx32 in granulosa and in thecal cells, suggests that follicular cells of the primate ovary utilize GJs with different Cx compositions. Identification of the factors regulating the presumed homo- and, perhaps, heterotypic GJ channels, will lead to a better understanding of cell-cell interactions affecting follicular growth, ovulation and atresia.